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Clinical Infection & Immunity

Original Article

Volume 5, Number 3, September 2020, pages 59-65

Genotypic Resistance Profile to Antiretrovirals in Children and Adolescents With Human Immunodeficiency Virus in a Reference Hospital in the Southern Region of Brazil

The human immunodeficiency virus type 1 (HIV-1) in the pediatric population comprises children living with the retrovirus, characterized as carriers with two detectable viral loads above 5,000 copies/mL [1], and who were mostly contaminated by vertical transmission peripartum [1, 2], with the main risk factor for transmission via this route being the detectable maternal viral load [1, 3].

Early use of antiretroviral therapy (ART) in children aims to improve growth and development, reducing morbidity and mortality associated with the virus. When at ideal plasma concentrations and optimal adherence to treatment, it is able to decrease infections associated with immune damage and reduce the replication of retroviruses. This is an important characteristic in this population, as pediatric patients have a higher viral load and may take longer to reach undetectable levels [1, 4]. These effects control the progress of the disease and decrease the chance of mutations and resistance [5-8].

On the other hand, it is known about the difficulty of adhering to therapy in this age group, and the main factors that lead to adherence problems are the long period of treatment, since children will use ART for much longer than one adult who contracts HIV, dependence on the guardian, side effects and lack of palatability of the drugs [5, 7, 9].

Partial adherence is one of the main causes of therapeutic failure [10], as it favors the emergence of HIV-1 resistance mutations. This is due to the high intrinsic mutational capacity of the virus, when it is able to easily incorporate the mutation into its genes [8], associated with viral selectivity caused by antiretroviral, and the main genotypic changes are in reverse transcriptase and viral protease [1, 6, 11-13].

Viral resistance leads to therapeutic failure [1], which is identified when clinical, viral and immunological parameters worsen, with the clinicians being more delayed [8]. When failure occurs, genotyping testing is necessary to guide therapeutic rescue [1, 8, 14, 15].

Some Brazilian studies carried out with children [5, 6] demonstrate that the main resistance mutations are associated with nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). These data are worrying, since these classes are instituted as initial therapy for the pediatric population that living with HIV.

The analysis of resistance profiles in the pediatric population guides medical and public policy decisions for choosing the best medication, aiming at satisfactory therapeutic results that reduce the morbidity and mortality associated with HIV. From the studies published in Brazil, there is no knowledge about the profile of resistance to antiretrovirals (ARVs) in the state of Santa Catarina, one of the main pockets of subtype C. With this, it is intended to assess the profile of genotypic resistance of children and adolescents with HIV in a Reference Hospital in the southern region of Brazil.

It is characterized as a descriptive observational study with a cross-sectional design. The collection was carried out at Day Hospital of Joana de Gusmao Children’s Hospital (JGCH), which is a state reference for the care of children and adolescents with HIV [16]. The data were collected from the physical and electronic medical records of the patients being monitored and the socioeconomic aspects were collected through interviews.

All children and/or adolescents being monitored at the referred hospital were evaluated during the period from July 2016 to February 2017. The study included those who met the diagnostic criteria for HIV infection, from 6 months to 14 years 11 months and 29 days, of both sexes, with genotyping. The patients or their guardians were approached at Day Hospital during the medical consultation, invited to participate in the research, and their consents were given by signing the informed consent forms (ICFs) when necessary.

The demographic variables analyzed in the current study were age, genre and ethnicity. Regarding clinical and laboratory characteristics, cluster of differentiation 4 (CD4)⁺ (< 350 cells/mm³, 350 - 500 cells/mm³ and > 500 cells/mm³) were analyzed, Viral Load (0 - 400 copies/ mL, 401 - 1,000 copies/mL, 1,001 -10,000 copies/mL, 10,001 - 100,000 copies/mL, > 100,000 copies/mL), use of ART, time on ART (in complete years), therapeutic regimen (dual therapy (NRTI + PI), triple therapy (NRTI + NNRTI), triple therapy (NRTI + NNRTI + PI), triple therapy (NRTI + PI + integrase inhibitor (INI)), therapeutic failure (yes, no), viral subtype, total mutations found and by ARV class (absolute values and identification of mutations) and susceptibility to ARV’s (sensitivity, resistance, intermediate resistance and possible resistance).

A database was created in a Microsoft Excel spreadsheet, later exported to the IBM SPSS Statistics 18.0^® software, to perform statistical analysis. The results were summarized as absolute numbers and percentages for nominal variables, mean and standard deviation and minimum and maximum values for numerical variables.

This study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki Declaration. The study was submitted to and approved by the research ethics committee involving human beings of the University of the South of Santa Catarina (UNISUL) under CAAE 56787316.9.0000.5369 and JGCH ethics committee under CAAE 56787316.9.3001.5361.

Forty-four children and adolescents diagnosed with HIV infection, who have genotyping tests, were evaluated. The mean age of the patients was 7.95 ± 3.73 years, with a minimum of 1 and a maximum of 14 years. Of these, there was a predominance of females, of non-white ethnicity, from the age groups of 5 to 11 years of age (Table 1).

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Table 1. Demographic Characterization of Children and Adolescents With HIV Treated at a Reference Hospital in Santa Catarina, 2016 - 2017

It was found that 77.3% had an CD4⁺ count above 500 cells/mm³, most with a viral load between 0 and 400 copies. Of the group tested, 90.9% of the patients were using some type of antiretroviral, with 67.5% predominantly using a triple regimen with the association of NRTI with PI. According to the information obtained from the medical records, 85.0% had no therapeutic failure (Table 2).

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Table 2. Clinical and Laboratory Characterization of Children and Adolescents with HIV Treated at a Reference Hospital in Santa Catarina, 2016 - 2017

Among the children and/or adolescents evaluated, only 21 (47.72%) presented some type of resistance mutation, with an average of 3.43 ± 2.27 mutations. Of these, 61.9% were identified in NRTI, 80.95% for NNRTI and 14.29% for PI.

Among the patients who presented resistance, the mean mutations in the NRTIs were 2.61 ± 2.10 (minimum of 1 and maximum of 6), in the NNRTIs it was 1.94 ± 0.55 (minimum of 1 and maximum of 3) and in the PIs it was 1.67 ± 1.15 (minimum of 1 and maximum of 3).

In relation to HIV mutations by classes of antiretroviral drugs, the most frequently identified NRTIs were 211K (33.3%), 214F (28.3%) and 184V (10.1%). In NNRTIs, the most found were K103N (34.8%), Y181C (13%), V179D (13%), and G190A (13%) (Table 3).

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Table 3. HIV Mutations Identified in NRTI and NNRTI in Children and Adolescents Treated at a Reference Hospital in Santa Catarina, 2016 - 2017

As for other polymorphisms in reverse transcriptase, 245Q (8.2%), 200A (7.2%), 48T (6.9%) and 177E (5.5%) stood out (Table 4).

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Table 4. HIV Mutations Identified as Other Polymorphisms in Reverse Transcriptase in Children and Adolescents Treated at a Reference Hospital in Santa Catarina, 2016 - 2017

The most frequent mutations associated with PI were 93L (13.3%), 69K (12.9%), 36I (11.3%), 89M (10.9%) and 15V (10.5%) (Table 5).

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Table 5. HIV Mutations Associated With PI in Children and Adolescents Treated at a Reference Hospital in Santa Catarina, 2016 - 2017

In the other protease polymorphisms class, 37K (25.6%), 36M (21.7%), 41N (17.4%), 14R (7.4%), 12A (4.1%) and 19I (4.1%) were the most representative.

The duration of use of ART had an average of 4.81 ± 3.65 years, with a minimum of 1 and a maximum of 13 years. The most frequent viral subtype was C in 90.9% of patients.

Figure 1 shows the susceptibility to ARV’s in children and adolescents with HIV. Resistance to lamivudine (3TC) (23.3%), zidovudine (ZDV) + 3TC (20.0%), efavirenz (EFV) (31.8%) and nevirapine (NVP) (36.4%) and intermediate resistance to didanosine (ddI) (20.5%) were observed. The sensitivity was 100% for atazanavir/ritonavir (ATVr), darunavir/ritonavir (DRVr), indinavir/ritonavir (IDVr), lopinavir/ritonavir (LPVr), tipranavir/ritonavir (TPVr) and fosamprenavir (FPV).

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Figure 1. Susceptibility to antiretrovirals in children and adolescents treated at a reference hospital in Santa Catarina, 2016 - 2017. NRTI: nucleoside reverse transcriptase inhibitor, lamivudine (3TC), abacavir (ABC), zidovudine (ZDV), tenofovir (TDF), didanosine (ddI), stavudine (d4T); NNRTI: non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), etravirine (ETV), nevirapine (NVP); PI: protease inhibitor, atazanavir/ritonavir (ATVr), darunavir/ritonavir (DRVr), fosamprenavir/ritonavir (FPVr), lopinavir/ritonavir (LPVr), saquinavir/ritonavir (SQVr), tipranavir/ritonavir (TPVr), atazanavir (ATV), fosamprenavir (FPV), ritonavir (RTV), saquinavir (SQV), nelfinavir (NFV), indinavir (IDV), indinavir/ritonavir (IDVr).

Given of extensive research in the literature and as far as is known, this is the first study to assess genotypic resistance in children and adolescents with HIV due to vertical transmission in southern Brazil.

We demonstrated that in Santa Catarina the viral subtype C (90.9%) prevails in the researched population. This is probably due to the fact that HIV-1C is more frequent in heterosexual adults [17] and pregnant women [18], thus enabling vertical transmission. Another hypothesis is that the state receives a large number of tourists because it has port cities and borders Argentina, enabling the possibility of several recombinants of viral subtypes [18-20]. The findings corroborate with other studies carried out in adults [4, 17, 19], where the South of Brazil is a pocket of this subtype of HIV-1 [21] with the highest prevalence in the Americas [19] and is worrying when it differs from the rest of the country because it is known that the classes non-B has been associated with less susceptibility to drugs and increases the chance of resistance in children while using ART [22-24].

The average time of use of antiretrovirals was 4.81 years, demonstrating that the diagnosis and drug introduction occurred early, since the average age found among the respondents was 7.95 years. Studies have demonstrated the possibility of an accumulated incidence of resistance and virologic failure when ART has been in use for more than 4 - 5 years [11, 24-27] and research with African children [28] has shown drug resistance in patients with 12 months of treatment. In the present study, although it is not possible to establish at which time of treatment genotyping was performed, we assume that the transmitted resistance was responsible for cases of early virologic failure. The maximum time of use of ART observed was 13 years. This can be worrisome because, since children and adolescents surveyed will use ART for much longer than adults, there is a greater risk of resistance in the population surveyed.

Of the 78 children and/or adolescents evaluated, only 44 had undergone genotyping, which is outside the recommendations of the Ministry of Health, which recommends that this test be performed pre-treatment or in the presence of therapeutic failure for all children and adolescents [1]. Of these children, 21 had at least one resistance mutation, with an average of 3.43 ± 2.27. This fact is not different from what we expected, since Brazil is one of the countries with the highest number of resistances [24]. This is believed to occur because the treatment of pediatric HIV carriers has been available free of charge by the Unified Health System (UHS) since 1996 [29], allowing for a long time of exposure and facilitating changes in acquired resistance. It is known that gestational exposure to ineffective plasma concentrations of antiretrovirals, due to the irregular use of ART by the mother during pregnancy, also contribute to increased resistance [12, 30].

The difficulty in adhering to therapy in this age group can also contribute to a greater number, since partial adherence is one of the main causes of therapeutic failure [10], favoring the emergence of more complex resistance mutations in the pediatric population [5, 31].

The antiretrovirals that showed the highest resistance by class were EFV (31.8%) and NVP (36.4%). EFV and NVP are part of the first line of treatment at least since 2014 and the prevention of mother-to-child transmission (PMTCT), a fact that may explain the high levels of resistance found. This data converges with studies carried out with children and adolescents in Bahia [5].

The major mutations associated with NRTIs were K103N, Y181C and G190A, in line with several other studies [5, 28, 31, 32] and are more frequently associated with PMTCT [31, 32].

In 2016, the year in which the data were collected, the use of dual therapy consisting of an NRTI and an NNRTI was recommended (EFV or NVP depending on the age group and body weight) or as a second line the triple therapy composed of two NRTIs and PI. Of the children and adolescents surveyed, 97.5% were on triple therapy, with 67.5% using second-line regimens (NRTI + PI) and 25% first-line regimens (NRTI + NNRTI). Four patients did not use any type of antiretroviral and three were not in compliance with the recommendations of the Ministry of Health [33] because they did not fit the proposed scheme.

Currently, drugs registered as the first line are composed of two NRTIs associated with an INI (which depends on the age group) and the drug alternative is composed of two NRTIs plus one NNRTI [1]. EFV and NVP, drugs that showed the most resistance in this study, remain an option to rescue the treatment of children and adolescents and were considered the first line of treatment according to the protocol of the year surveyed [33].

The Y181C mutation has been associated with patients exposed to NVP doses [28]. ZDV + NVP is currently recommended in newborns of mothers infected with a viral load above 1,000 copies/mL in the third trimester and/or who did not use ART during pregnancy [1].

Of the major mutations associated with NRTI, M184V was the most frequent (10.1%). This mutation is the most common mutation found for this class and despite giving high rates of resistance to 3TC, it decreases viral replication and increases susceptibility to TDF and ZDV [34]. 211K and 214F were more frequent but are not considered major mutations.

Mutations for thymidine analogues were found in 19% of the patients, 4% of them being D67N. These are responsible for reducing susceptibility to NRTI [34] and its appearance increases proportionally to the time of use of antiretrovirals [35, 36] and is probably associated with the use of ZDV recommended in past protocols as the first line of treatment. No multiple drug resistance (MDR) mutations were found for this class.

No major mutations associated with PI were found, and only one accessory V11I mutation was present in 0.4%. This is associated with the previous use of darunavir [34]. This result was expected because PIs are drugs with a high genetic barrier and although they are not part of the first line of treatment in our country, they were contained in most of the researched schemes.

As for viral load, 38.6% had a count with more than 1,000 copies/mL of viral particles, suggesting that factors such as low adherence incurring virologic failure or acquired and transmitted resistance may be present. Viral suppression is essential for successful treatment [1].

There are few studies that demonstrate the profile of children and adolescents with HIV in Brazil. We found a prevalence of females, non-white ethnicity, with a mean age of 7.95 ± 3.73 years, CD4⁺ above 500 cells/mm³, using triple ART containing 2NRTI + 1PI. The predominant viral subtype was C, with 47.72% having some type of drug resistance mutations. The most frequent major mutations by class were M184V and K103N. These mutations were not found for PIs. The antiretrovirals that showed the most resistance were EFV and NVP. We concluded with this research that the drugs used as the first line of treatment for the protocol of the year researched and as a redemption option in the current one, have high rates of resistance in the researched population. We need to pay attention to the possibilities of ART in children since the options are limited and children will use this class of drugs for a longer period of time than adults, increasing the chance of virologic failure and acquired resistance. We demonstrate the need to perform more pre-treatment genotyping as several resistance mutations have been found that decrease susceptibility to the most commonly used drugs.

This study has some limitations. Unfortunately, it was not possible to accurately establish patients in therapeutic failure, since these data were not always found in the medical records. In addition, it was not possible to assess adherence to treatment or gestational and perinatal exposure to antiretrovirals.

Acknowledgments

We are grateful to those responsible for patients for their willingness to answer questions. We would like to thank the sector doctors and Day Hospital employees for their collaboration with the physical and electronic medical records.

Financial Disclosure

This study received no funding source.

Conflict of Interest

The authors declare that they have no conflict of interest.

Informed Consent

The patients or their guardians invited to participate in this research were given the consents by signing the informed consent forms (ICFs).

Authors Contributions

SDN conceptualized and conducted the research, which provided the basis for the article. SDN supported the collection of data in the field, as well as in the laboratory, and data analysis, assembly of tables, interpretation. SDN correlated what was found with what was in the literature for the preparation of this manuscript. MRdN supported the planning of the research, preparation of the questionnaire for data collection and the collection of the same data going to the field. MRdN participated in the interpretation and analysis of the data obtained. ADS supported the study planning, guided the data collection in the field and part of the laboratory analysis, as well as supported the data analysis and writing the article.

Data Availability

The authors declare that the data supporting the findings of this study are available within the article.

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